This invention relates to an apparatus and process for effecting a plurality of sequential chemical syntheses in a plurality of reaction modules each containing a solid support.
Instrumentation for the automatic synthesis of peptides has been available since 1964, when Merrifield described the first automated solid-phase peptide synthesizer. Since that time Merrifield-type peptide synthesizers employing Merrifield chemistry (Boc-protected amino acids, polystyrene based synthesis resins, deprotection with trifluoroacetic acid, cleavage of peptide from resin with hydrogen fluoride) have come into wide use. More recently, the so-called Fmoc method of synthesis, Atherton et al. (1979) Bioorg. Chem 8, 351, (Fmoc-protected amino acids, protective acrylamide-silica gel based synthesis supports, deprotection with alkaline reagents, cleavage of peptides from supports with trifluoroacetic acid) have become popular. One characteristic that distinguishes the Merrifield and the Fmoc syntheses is that the former requires that reactions be carried out in a shaken reaction cell, whereas in the latter case the support can be packed into a column and reagents pumped through. The flow-through synthesis capabilities of the Fmoc method give it several advantages, which is one reason it has become increasingly popular in recent years.
Also in recent years there has been an increase in demand for small peptides of similar structure. One type of need is for epitope mapping of proteins, i.e., a search for the small regions (6-12 amino acids) of proteins that are antigenic sites for binding of antibodies; or immunogenic sites, which stimulate the immune response. Immunogenic peptides have the potential for use in making vaccines. One way to search for these sites in a protein containing, for example, 200 amino acids, is to synthesize a set of approximately 200 overlapping hexapeptides, each differing from its neighbor by a single amino acid. Other applications are the synthesis of analogs of a biologically active peptide, either to find a more active peptide, or to determine which amino acids are responsible for activity, by systematic variation of the sequence. Synthesis of such large numbers of peptides one by one, such as those set forth below, even using a machine, is very time consuming.
ABCDEFGHIJKLMNOP ABCDEFG ABCDEFG PA1 ABCDE XBCDEFG AXCDEFG PA1 BCDEF AXCDEFG AYCDEFG PA1 CDEFG ABXDEFG AWCDEFG PA1 DEFGH ABCXEFG AZCDEFG PA1 EFGHI ABCDXFG AQCDEFG
There have been several methods and devices described to speed up this process. One of the first is the "teabag" method of Houghton (1985), Proc. Natl. Sci. USA 82, pg. 531 where synthesis is carried out on resins in small porous bags, which are soaked in solutions of the appropriate activated amino acid. Several bags can be placed in a single reaction vessel, and by proper "mixing and matching", several similar peptides can be synthesized (on a 50-100 umole scale) simultaneously. This process has not been automated, however. Another method is the "dipstick method" of Geysen et al., (1985) Proc. Natl. Acad. Sci. USA 82, 178-182 wherein very small quantities (1 umole) of peptide are synthesized on small polypropylene rods by dipping the rods into the appropriate solutions. The quantities that can be made are very small, and the process is not automated. Other multiple peptide synthesis systems are DuPont's RaMPS system, which is manual, and the method of Schnorrenberg et al. (1989), Tetrahedron 45, pgs 7759-7764. The latter device uses a robot arm to deliver reagents to synthesis support resins in wells of a micro-titre plate, and is purported to be capable of synthesizing 96 peptides at once.
It has also been proposed in U.S. Pat. No. 4,728,502 to provide an apparatus for effecting a series of sequential chemical reactions to form similar long chain molecules such as nucleic acids or peptides. The apparatus comprises a set of stacked plates, rotatable with respect to one another. Ports and reaction vessels containing a solid support are provided in each plate, so that reactant flow can be controlled through the plates to the desired reactor vessels. This process is undesirable since the flow of reactants is limited to only a small number of reaction vessels at a given set of plate positions and because reactant leakage can occur between the plates.
Accordingly, it would be desirable to provide a method and apparatus for simultaneously synthesizing in an automated fashion, in varying amounts, a plurality of similar chemical products such as peptides which are synthesized by sequentially adding molecules such as an amino acid to a chain of such molecules to form peptides.